Conotoxins I

ABSTRACT

Substantially pure conotoxins are provided which inhibit synaptic transmissions at the neuromuscular junctions and which are useful both in vivo and in assays because they specifically target particular receptors, such as the acetyl-choline receptor, and ion channels. The peptides are of such length that they can be made by chemical synthesis. They also may be made using recombinant DNA techniques, and the DNA encoding such conotoxins having pesticidal properties can be incorporated as plant defense genes into plant species of interest.

This is a division of application Ser. No. 08/084,848, filed on Jun. 29,1993, now U.S. Pat. No. 5,432,155.

This invention was made with Government support under Grant Nos.GM-22737 and AM-26741, awarded by the National Institute of Health. TheGovernment has certain rights in this invention.

This invention relates to relatively short peptides, and moreparticularly to peptides between about 16 and about 46 residues inlength, which are naturally available in minute amounts in the venom ofthe cone snails and which may include one or more cyclizing disulfidelinkages.

BACKGROUND OF THE INVENTION

Mollusks of the genus Conus produce a highly toxic venom which enablesthem to carry out their unique predatory lifestyle. Prey are immobilizedby the venom which is injected by means of a highly specialized venomapparatus, a disposable hollow tooth which functions both in the mannerof a harpoon and a hypodermic needle.

Few interactions between organisms are more striking than those betweena venomous animal and its eaveaomated victim. Venom may be used as aprimary weapon to capture prey or as a defense mechanism. These venomsdisrupt essential organ systems in the envenomated animal, and many ofthese venoms contain molecules directed to receptors and ion channels ofneuromuscular systems.

The predatory cone snails (Conus) have developed a unique biologicalstrategy. Their venom contains relatively small peptides that aretargeted to various neuromuscular receptors and may be equivalent intheir pharmacological diversity to the alkaloids of plants or secondarymetabolites of microorganisms. Many of these peptides are among thesmallest nucleic acid-encoded translation products having definedconformations, and as such they are somewhat unusual because peptides inthis size range normally equilibrate among many conformations forproteins having a fixed conformation are generally much larger.

The cone snails that produce these toxic peptides, which are generallyreferred to as conotoxins or conotoxin peptides, are a large genus ofvenomous gastropods comprising approximately 500 species. All cone snailspecies are predators that inject venom to capture prey, and thespectrum of animals that the genus as a whole can envenomate is broad. Awide variety of hunting strategies are used; however, every Conusspecies uses fundamentally the same basic pattern of envenomation.

The major paralytic peptides is these fish-hunting cone venoms were thefirst to be identified and characterized. In C. geographus venom, threeclasses of disulfide-rich peptides were found: the α-conotoxins (whichtarget and block the nicotinic acetylcholine receptors); theμ-conotoxins (which target and block the skeletal muscle Na⁺ channels);and the Ω-conotoxins (which target and block the presynaptic neuronalCa²⁺ channels). However, there are multiple homologs in each toxinclass; for example, at least five different Ω-conotoxins are present inC. geographus venom alone. Considerable variation in sequence isevident, and when different Ω-conotoxin sequences were first compared,only the cysteine residues that are involved in disulfide bonding andone glycine residue were found to be invariant. Another class ofconotoxins found in C. geographus venom is that referred to as theconantokins which cause sleep in young mice and hyperactivity in oldermice and are targeted to the NMDA receptor. Each cone venom appears tohave its own distinctive group or signature of different conotoxinsequences.

Many of these peptides have now become fairly standard research tools inneuroscience. The μ-conotoxins, because of their ability topreferentially block muscle but not ax nal Na⁺ channels, are convenienttools for immobilizing skeletal muscle without affecting ax nal orsynaptic events. The Ω-conotoxins have become standard pharmacologicalreagents for investigating voltage-sensitive Ca²⁺ channels and are usedto block presynaptic termini and neurotransmitter release. TheΩ-conotoxin GVIA from C. geographus venom, which binds to neuronalvoltage-sensitive Ca²⁺ channels, is an example of such. The affinity(K_(d)) of Ω-conotoxin GVIA for its high-affinity targets issub-picomolar; it takes more than 7 hours for 50% of the peptide todissociate. Thus the peptide can be used to block synaptic transmissionvirtually irreversibly because it inhibits presynaptic Ca²⁺ channels.However, Ω-conotoxin is highly tissue-specific. In contrast to thestandard Ca²⁺ channel-blocking drugs (e.g. the dihydropyridines, such asnifedipene and nitrendipene, which are widely used for angins andcardiac problems), which can bind Ca²⁺ channels is smooth, skeletal, andcardiac muscle as well as neuronal tissue, Ω-conotoxins generally bindonly to a subset of neuronal Ca²⁺ channels, primarily of the N subtype.The discrimination ratio for Ω-conotoxin binding to voltage-sensitiveCa²⁺ channels in neuronal versus nonneuronal tissue (e.g. skeletal orcardiac muscle) is greater than 10⁸ in many cases.

Additional conotoxin peptides having these general properties continueto be sought.

SUMMARY OF THE INVENTION

The present invention provides a group of bioactive conotoxin peptideswhich are extremely potent inhibitors of synaptic transmission at theneuromuscular junction and/or which are targeted to specific ionchannels. They are useful as pesticides, and many of them or closelyrelated analogs thereof are targeted to specific insects or other pests.Therefore, the DNA encoding such conotoxin peptides can beadvantageously incorporated into plants as a plant-defense gene toreader plants resistant to specified pests.

The conotoxin peptides have the formulae set forth hereinafter.Moreover, examination of the formulae shows an indication of two newclasses of conotoxin peptides in addition to those classes hereinbeforedescribed. Class A includes peptides SEQ ID NO:1 to NO:6; each has 6 Cysresidues which are interconnected by 3 disulfide linkages, with the 2Cys residues nearest the N-terminus being part of a sequence-Cys-Cys-Gly-. All 6 members have at least one 4Hyp residue and aC-terminus which appears to be amidated. There are 2 amino acid (AA)residues separating the 3rd and 4th Cys as numbered (from theN-terminus) and a single AA residue spacing the 4th Cys from the 5thCys. Moreover, the 2nd Cys is usually separated from the 3rd Cys byeither 6 or 7 AA residues in this class, whereas there can be from about3 to about 6 AA residues separating the 5th and 6th Cys residues. ClassB is exemplified by SEQ ID NO:7, wherein there is a central sequence of5 AA residues having 2 pairs of Cys residues flanking a center residuewhich is preferably Asn and wherein there are 2 additional pairs ofspaced-apart Cys residues located, respectively. N-terminally andC-terminally of this central sequence. SEQ ID NO:8 appears to be amember of the known class of α-conotoxins. SEQ ID NO:9 appears to be amember of the known class of μ-conotoxins. SEQ ID NO:10 and NO:11 may bemembers of the class of Ω-conotoxins. SEQ ID NO:12 appears to be amember of the class of conantokins characterized by the N-terminalsequence Gly-Glu-Gla-Gla, and SEQ ID NO:13 may be a member of aheretofore uncharacterized class which causes sluggish behavior. Theindividual formulae of these conotoxins are as follows:

-   -   Gly-Cys-Cys-Gly-Ser-Tyr-Pro-Asn-Ala-Ala-Cys-His-Pro-Cys-Ser-Cys-Lys-Asp-Arg-Xaa-Ser-Tyr-Cys-Gly-Gln        (SEQ ID NO:1) (J-020), wherein Xaa is 4Hyp (4-hydroxyproline)        and the C-terminus is amidated;    -   Glu-Lys-Ser-Leu-Val-Pro-Ser-Val-Ile-Thr-Thr-Cys-Cys-Gly-Tyr-Asp-Xaa-Gly-Thr-Met-Cys-Xaa-Xaa-Cys-Arg-Cys-Thr-Asn-Ser-Cys        (SEQ ID NO:2) (J-005) wherein Glu in the 1-position is pGlu, Xaa        is 4Hyp and the C-terminus is amidated; Ser in the 7-position        may be glycosylated;    -   Cys-Cys-Gly-Val-Xaa-Asn-Ala-Ala-Cys-Pro-Xaa-Cys-Val-Cys-Asn-Lys-Thr-Cys-Gly        (SEQ ID NO:3) (OB-34) wherein Xaa is 4Hyp and the C-terminus is        amidated;    -   Gly-Cys-Cys-Gly-Ser-Tyr-Xaa-Asn-Ala-Ala-Cys-His-Xaa-Cys-Ser-Cys-Lys-Asp-Arg-Xaa-Ser-Tyr-Cys-Gly-Gln        (SEQ ID NO:4) (J-019) wherein Xaa is 4Hyp and the C-terminus is        amidated;    -   Gly-Cys-Cys-Gly-Ser-Tyr-Xaa-Asn-Ala-Ala-Cys-His-Pro-Cys-Ser-Cys-Lys-Asp-Arg-Xaa-Ser-Tyr-Cys-Gly-Gln        (SEQ ID NO:5) (J-026) wherein Xaa is 4Hyp and the C-terminus is        amidated;    -   Cys-Cys-Gly-Val-Xaa-Asn-Ala-Ala-Cys-His-Xaa-Cys-Val-Cys-Lys-Asn-Thr-Cys        (SEQ ID NO:6) (OB-26) wherein Xaa is 4Hyp and the C-terminus is        amidated;    -   Gly-Xaa-Ser-Phe-Cys-Lys-Ala-Asp-Glu-Lys-Xaa-Cys-Glu-Tyr-His-Ala-Asp-Cys-Cys-Asn-Cys-Cys-Leu-Ser-Gly-Ile-Cys-Ala-Xaa-Ser-Thr-Asn-Trp-Ile-Leu-Pro-Gly-Cys-Ser-Thr-Ser-Ser-Phe-Phe-Lys-Ile        (SEQ ID NO:7) (J-029) wherein Xaa is 4Hyp; the C-terminus may        optionally be amidated;    -   Gly-Cys-Cys-Ser-His-Pro-Ala-Cys-Ser-Gly-Lys-Tyr-Gln-Xaa-Tyr-Cys-Arg-Xaa-Ser        (SEQ ID NO:8) (OB-20) wherein Xaa is and the C-terminus is        amidated;    -   His-Xaa-Xaa-Cys-Cys-Leu-Tyr-Gly-Lys-Cys-Arg-Arg-Tyr-Xaa-Gly-Cys-Ser-Ser-Ala-Ser-Cys-Cys-Gln        (SEQ ID NO:9) (J-021) wherein Xaa is 4Hyp;    -   Cys-Lys-Thr-Tyr-Ser-Lys-Tyr-Cys-Xaa-Ala-Asp-Ser-Xaa-Cys-Cys-Thr-Xaa-Gln-Cys-Val-Arg-Ser-Tyr-Cys-Thr-Leu-Phe        (SEQ ID NO:10) (J-010) wherein Xaa is Gla and the C-terminus is        amidated;    -   Ser-Thr-Ser-Cys-Met-Glu-Ala-Gly-Ser-Tyr-Cys-Gly-Ser-Thr-Thr-Arg-Ile-Cys-Cys-Gly-Tyr-Cys-Ala-Tyr-Phe-Gly-Lys-Lys-Cys-Ile-Asp-Tyr-Pro-Ser-Asn        (SEQ ID NO:11) (J-008);    -   Gly-Glu-Xaa-Xaa-Val-Ala-Lys-Met-Ala-Ala-Xaa-Leu-Ala-Arg-Xaa-Asn-Ile-Ala-Lys-Gly-Cys-Lys-Val-Asn-Cys-Tyr-Pro        (SEQ ID NO:12) (J-017) wherein Xaa is Gla (γ-carboxyglutmate);        and    -   Glu-Ser-Glu-Glu-Gly-Gly-Ser-Asn-Ala-Thr-Lys-Lys-Pro-Tyr-Ile-Leu        (SEQ ID NO:13) (J-004), wherein Glu in the 1-position is pGlu        (pyroglutamic) and the C-terminus may be amidated; Thr may be        glycosylated.

Accordingly in one aspect, the invention provides conotoxin peptideshaving the general formula:Xaa₁-Cys-Cys-Gly-Xaa₂-Cys-Xaa₃-Xaa₄-Cys-Xaa₅-Cys-Xaa₆-Cys-Xaa₇-NH₂ (SEQID NO:14) wherein Xaa₁ is des-Xaa₁ or Gly orpGlu-Lys-Ser-Leu-Val-Pro-Ser-Val-Ile-Thr-Thr; Xaa₂ isSer-Tyr-Pro-Asn-Ala-Ala or Tyr-Asp-4Hyp-Gly-Thr-Met orVal-4Hyp-Asn-Ala-Ala or Ser-Tyr-4Hyp-Asn-Ala-Ala; Xaa₃ is His, 4Hyp orPro; Xaa₄ is Pro or 4Hyp; Xaa₅ is Ser, Arg or Val; Xaa₆ isLys-Asp-Arg-4Hyp-Ser-Tyr or Thr-Asn-Ser or Asn-Lys-Thr or Lys-Asn-Thr;and Xaa₇ is des-Xaa₇ or Gly or Gly-Gln.

In another aspect, the invention provides conotoxin peptides having 6Cys residues interconnected by 3 disulfide bonds, with the 2 Cysresidues nearest the N-terminus being part of the sequence Cys-Cys-Glyand with the 3rd, 4th and 5th residue being spaced apart by 2 residuesand 1 residue, respectively, said two residues being selected from His,Pro and 4Hyp, said single residue being Ser, Arg or Val and with theC-terminus being amidated, said conotoxin binding to the acetylcholinereceptor.

In yet another aspect, the invention provides conotoxin peptides having8 Cys residues interconnected by 4 disulfide bonds with the central 4Cys residues being part of the sequence Cys-Cys-Asn-Cys-Cys (SEQ IDNO:15), said conotoxin causing immediate paralysis where administeredintercranially to laboratory mice.

These peptides, which are generally termed conotoxins, are sufficientlysmall to be chemically synthesized. General chemical syntheses forpreparing the foregoing conotoxins are described hereinafter along withspecific chemical syntheses of several conotoxins and indications ofbiological activities of these synthetic products. Various of theseconotoxins can also be obtained by isolation and purification fromspecific conus species using the techniques described in U.S. Pat. No.4,447,356 (May 8, 1984), the disclosure of which is incorporated hereinby reference.

Many of these conotoxin peptides are extremely potent inhibitors ofsynaptic transmission at the neuromuscular junction, while at the sametime lacking demonstrable inhibition of either nerve or muscle actionpotential propagation. They are considered useful to relax certainmuscles during surgery.

The activity of each of these conotoxin peptides is freely reversibleupon dilution or removal of the toxin from the affected muscle.Moreover, toxicity of the cyclic peptides is generally destroyed byagents which disrupt disulfide bonds in the cyclic conotoxins,suggesting that correct disulfide bonding appears essential forbiological activity; however, correct folding and/or rearrangement of aconotoxin may occur in vivo so that in some cases the linear peptide maybe administered for certain purposes. In general, however, the syntheticlinear peptides fold spontaneously when exposed to air-oxidation at coldroom temperatures to create the correct disulfide bonds to conferbiological activity, and such processing is accordingly preferred. Theconotoxins exhibit activity on a wide range of vertebrate animals,including humans, and on insects, and many are useful to reversiblyimmobilize a muscle or group of muscles in humans or other vertebratespecies. Many of these conotoxins and derivatives thereof are furtheruseful for detection and measurement of acetylcholine receptors andother specific receptors which are enumerated hereinafter with respectto various particular peptides.

Many of these conotoxin peptides are also useful in medical diagnosis.For example, an immunoprecipitation assay with radiolabeled Ω-conotoxincan be used to diagnose the Lambert-Eaton myasthenic syndrome, which isa disease in which autoimmune antibodies targeted to endogenous Ca²⁺channels are inappropriately elicited, thereby causing muscle weaknessand autonomic dysfunction.

Various of these conotoxin peptides are further useful for the treatmentof neuromuscular disorders and for rapid reversible immobilization ofmuscles in vertebrate species, including humans, thereby facilitatingthe setting of fractures and dislocations. These conotoxins generallyinhibit synaptic transmission at the neuromuscular junction and bondstrongly to the acetylcholine receptor of the muscle end plate, and manyare therefore especially suitable for detection and assay ofacetylcholine receptors. Such measurements are of particularsignificance in clinical diagnosis of myasthenia gravis, and various ofthese conotoxins, when synthesized with a radioactive label or as afluorescent derivative, provide improved quantitation and sensitivity inacetylcholine receptor assays.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

Although the conotoxins can be obtained by purification from theenumerated cone snails, because the amounts of conotoxins obtainablefrom individual snails are very small, the desired substantially pureconotoxins are best practically obtained in commercially valuableamounts by chemical synthesis. For example, the yield from a single conesnail may be about 10 micrograms or less of conotoxin. By substantiallypure is meant that the peptide is present in the substantial absence ofother biological molecules of the same type; it is preferably present inan amount at least about 85% by weight and more preferably at leastabout 95% of such biological molecules of the same type which arepresent, i.e. waters, buffers and innocuous small molecules may bepresent. Chemical synthesis of biologically active conotoxin peptidedepends of course upon correct determination of the amino acid sequence,and these sequences have now been determined and are set forth in thepreceding summary.

Many of the conotoxins have approximately the same level of activity,and comparison of them suggests a reasonable tolerance for substitutionnear the carboxy terminus of these peptides. Accordingly, equivalentmolecules can be created by the substitution of equivalent residues inthis region, and such substitutions are useful to create particularinvertebrate-specific conotoxins.

Cysteine residues are present in a majority of these conotoxins, andseveral of the conotoxins disclosed herein exhibit similar disulfidecross-linking patterns to that of erabutoxin, a known protein toxin ofsea snake venom. The fact that biological activity of these particularcompounds is destroyed by agents which break disulfide bonds, such assodium borohydride or β-mercaptoethanol, indicates that a specificfolded configuration induced by disulfide crosslinks, is essential forbioactivity of these particular conotoxins. It has been found thatair-oxidation of the linear peptides for prolonged periods under coldroom temperatures results in the creation of a substantial amount of thebioactive, disulfide-linked molecules. Therefore, the preferableprocedure for making these peptides is to oxidize the linear peptide andthen fractionate the resulting product, using reverse-phase highperformance liquid chromatography (HPLC) or the like, to separatepeptides having different linked configurations. Thereafter, either bycomparing these fractions with the elution of the native material or byusing a simple assay, the particular fraction having the correct linkagefor maximum biological potency is easily determined. It is also foundthat the linear peptide, or the oxidized product having more than onefraction, can sometimes be used for in vivo administration, because thecross-linking and/or rearrangement which occurs in vivo has been foundto create the biologically potent conotoxin molecule; however, becauseof the dilution resulting from the presence of other fractions of lessbiopotency, a somewhat higher dosage may be required.

These conotoxins disclosed herein generally inhibit synaptictransmission at the neuromuscular junction by binding the acetylcholinereceptor at a muscle end plate. A particularly useful characteristic ofa number of these conotoxins is their high affinity for particularmacromolecular receptors, accompanied by a narrow receptor-targetspecificity. A major problem in medicine results from side effects whichdrugs very often exhibit, some of which are caused by the drug bindingnot only to the particular receptor subtype that renders therapeuticvalue, but also to closely related, therapeutically irrelevant receptorsubtypes which can often cause undesirable physiological effects. Incontrast to most drugs, these conotoxins generally discriminate amongclosely related receptor subtypes.

The peptides are synthesized by a suitable method, such as byexclusively solid-phase techniques, by partial solid-phase techniques,by fragment condensation or by classical solution couplings. Theemployment of recently developed recombinant DNA techniques may be usedto prepare these peptides, particularly the longer ones containing onlynatural amino acid residues which do not require post-translationalprocessing steps.

In conventional solution phase peptide synthesis, the peptide chain canbe prepared by a series of coupling reactions in which the constituentamino acids are added to the growing peptide chain in the desiredsequence. The use of various N-protecting groups, various couplingreagents, e.g., dicyclohexylcarbodiimide or carbonyldimidazole, variousactive esters, e.g., esters of N-hydroxyphthalimide orN-hydroxy-succinimide, and the various cleavage reagents, to carry outreaction in solution, with subsequent isolation and purification ofintermediates, is well known classical peptide methodology. Classicalsolution synthesis is described in detail in the treatise “Methoden derOrganischen Chemie (Houben-Weyl): Synthese von Peptiden”, E. Wunsch(editor) (1974) Georg Thieme Verlag, Stuttgart, W. Ger. Techniques ofexclusively solid-phase synthesis are set forth in the textbook“Solid-Phase Peptide Synthesis”, Stewart & Young, Freeman & Co., SanFrancisco, 1969, and are exemplified by the disclosure of U.S. Pat. No.4,105,603, issued Aug. 8, 1978 to Vale et al. The fragment condensationmethod of synthesis is exemplified in U.S. Pat. No. 3,972,859 by U.S.Pat. No. 3,842,067 (Oct. 15, 1974) and U.S. Pat. No. 3,862,925 (Jan. 28,1975).

Common to such chemical syntheses is the protection of the labile sidechain groups of the various amino acid moieties with suitable protectinggroups which will prevent a chemical reaction from occurring at thatsite until the group is ultimately removed. Usually also common is theprotection of an alpha-amino group on an amino acid or a fragment whilethat entity reacts at the carboxyl group, followed by the selectiveremoval of the alpha-amino protecting group to allow subsequent reactionto take place at that location. Accordingly, it is common that, as astep in such a synthesis, an intermediate compound is produced whichincludes each of the amino acid residues located in its desired sequencein the peptide chain with appropriate side-chain protecting groupslinked to various of the residues having labile side chains.

As far as the selection of a side chain amino protecting group isconcerned, generally one is chosen which is not removed duringdeprotection of the α-amino groups during the synthesis. However, forsome amino acids, e.g. His, protection is not generally necessary. Inselecting a particular side chain protecting group to be used in thesynthesis of the peptides, the following general rules are followed: (a)the protecting group preferably retains its protecting properties and isnot split off under coupling conditions (b) the protecting group shouldbe stable under the reaction conditions selected for removing theα-amino protecting group at each step of the synthesis, and (c) the sidechain protecting group must be removable, upon the completion of thesynthesis containing the desired amino acid sequence, under reactionconditions that will not undesirably alter the peptide chain.

It should be possible to prepare many, or even all, of these peptidesusing recombinant DNA technology; however, when peptides are not soprepared, they are preferably prepared using the Merrifield solid phasesynthesis, although other equivalent chemical syntheses known in the artcan also be used as previously mentioned. Solid-phase synthesis iscommenced from the C-terminus of the peptide by coupling a protectedα-amino acid to a suitable resin. Such a starting material can beprepared by attaching an α-amino-protected amino acid by an esterlinkage to a chloromethylated resin or a hydroxymethyl resin, or by anamide bond to a benzhydrylamine (BHA) resin or paramethylbenzhydrylamine(MBHA) resin. The preparation of the hydroxymethyl resin is described byBodansky et al., Chem. Ind. (London) 38, 1597-98 (1966).Chloromethylated resins are commercially available from Bio RadLaboratories, Richmond, Calif. and from Lab. Systems, Inc. Thepreparation of such a resin is described by Stewart et al., “Solid PhasePeptide Synthesis”, supra. BHA and MBHA resin supports are commerciallyavailable and are generally used when the desired polypeptide beingsynthesized has an unsubstituted amide at the C-terminus. Thus, solidresin supports may be any of those known in the art, such as one havingthe formulae: —O—CH₂-resin support, —NH BHA resin support or —NH—MBHAresin support. When the unsubstituted amide is desired, use of a BHA orMBHA resin is preferred, because cleavage directly gives the amide. Incase the N-methyl amide is desired, it can be generated from an N-methylBHA resin. Should other substituted amides be desired, the teaching ofU.S. Pat. No. 4,569,967 can be used, or should still other groups thanthe free acid be desired at the C-terminus, it may be preferable tosynthesize the peptide using classical methods as set forth in theHouben-Weyl text.

The C-terminal amino acid, protected by Boc and by a side-chainprotecting group, if appropriate, can be first coupled to achloromethylated resin according to the procedure set forth in ChemistryLetters, K. Horiki et al. 165-168 (1978), using KF in DMF at about 60°C. for 24 hours with stirring, when a peptide having free acid at theC-terminus is to be synthesized. Following the coupling of theBOC-protected amino acid to the resin support, the α-amino protectinggroup is removed, as by using trifluoroacetic acid (TFA) in methylenechloride or TFA alone. The deprotection is carried out at a temperaturebetween about 0° C. and room temperature. Other standard cleavingreagents, such as HCl in dioxane, and conditions for removal of specificα-amino protecting groups may be used as described in Schroder & Lubke,“The Peptides”, 1 pp 72-75, Academic Press (1965).

After removal of the α-amino protecting group, the remaining α-amino-and side chain-protected amino acids are coupled step-wise in thedesired order to obtain the intermediate compound defined hereinbefore,or as an alternative to adding each amino acid separately in thesynthesis, some of them may be coupled to one another prior to additionto the solid phase reactor. The selection of an appropriate couplingreagent is within the skill of the art. Particularly suitable as acoupling reagent is N,N′-dicyclohexyl carbodiimide (DCC).

The activating reagents used in the solid phase synthesis of thepeptides are well known in the peptide art. Examples of suitableactivating reagents are carbodiimides, such asN,N′-diisopropylcarbodiimide andN-ethyl-N′-(3-dimethylaminopropyl)carbodiimide. Other activatingreagents and their use in peptide coupling are described by Schroder &Lubke supra. in Chapter III and by Kapoor, J. Phar. Sci., 59, p 1-27(1970).

Each protected amino acid or amino acid sequence is introduced into thesolid phase reactor in about a twofold or more excess, and the couplingmay be carried out in a medium of dimethylformamide (DMF): CH₂Cl₂ (1:1)or in DMF or CH₂Cl₂ alone. In cases where incomplete coupling occurs,the coupling procedure is repeated before removal of the α-aminoprotecting group prior to the coupling of the next amino acid. Thesuccess of the coupling reaction at each stage of the synthesis, ifperformed manually, is preferably monitored by the ninhydrin reaction,as described by E. Kaiser et al., Anal. Biochem. 34, 595 (1970). Thecoupling reactions can be performed automatically, as on a Beckman 990automatic synthesizer, using a program such as that reported in Rivieret al. Biopolymers, 1978, 17, pp 1927-1938.

After the desired amino acid sequence has been completed, theintermediate peptide can be removed from the resin supported bytreatment with a reagent, such as liquid hydrogen fluoride, which notonly cleaves the peptide from the resin but also cleaves all remainingside chain protecting groups and also the α-amino protecting group atthe N-terminus if it was not previously removed to obtain the peptide inthe form of the free acid. If Met is present in the sequence, the Bocprotecting group is preferably first removed using trifluoroacetic acid(TFA)/ethanedithiol prior to cleaving the peptide from the resin with HFto eliminate potential S-alkylation. When using hydrogen fluoride forcleaving, one or more scavengers, such as anisole, cresol, dimethylsulfide, and methylethyl sulfide are included in the reaction vessel.

Cyclization of the linear peptide is preferably effected, as opposed tocyclizing the peptide while a part of the peptidoresin, to create bondsbetween Cys residues. To effect such a disulfide cyclizing linkage, thefully protected peptide can be cleaved from a hydroxymethylated resin ora chloromethylated resin support by ammonolysis, as is well known in theart, to yield the fully protected amide intermediate, which isthereafter suitably cyclized and deprotected; alternatively,deprotection as well as cleavage of the peptide from the above resins ora benzhydrylamine (BHA) resin or a methyl-benzhydrylamine (MBHA), cantake place at 0° C. with hydrofluoric acid (HF), followed byair-oxidation under high dilution conditions.

Thus, in one aspect, the invention also provides a method formanufacturing a synthetic conotoxin peptide of interest by carrying outthe following steps: (a) forming a peptide intermediate having thedesired amino acid residue sequence and at least one protective groupattached to a labile side chain of a residue such as Ser, Thr, Tyr, Asp,Glu, His, Cys, Arg or Lys and optionally having its C-terminus linked byan anchoring bond to resin support; (b) splitting off the protectivegroup or groups and any anchoring bond from the peptide intermediate toform a linear peptide; (c) creating a cyclizing bond between Cysresidues present in the linear peptide to create a cyclic peptide; and(d) if desired, converting the resulting cyclic peptide into a nontoxicsalt thereof. Particular side chain protecting groups and resin supportsare well known in the art and are disclosed in the earlier-referencedpatents.

In order to illustrate specific preferred embodiments of the inventionin greater detail, the following exemplary work is provided.

EXAMPLE 1

Conotoxin SEQ ID NO:1 (also referred to as J-020), having the chemicalformula:H-Gly-Cys-Cys-Gly-Ser-Tyr-Pro-Asn-Ala-Ala-Cys-His-Pro-Cys-Ser-Cys-Lys-Asp-Arg-4Hyp-Ser-Tyr-Cys-Gly-Gln-NH₂is synthesized by stepwise elongation from the carboxyl terminus, usingthe solid phase Merrifield peptide synthesis procedure. Operationaldetails of this general procedure, which are not set forth hereinafter,can be found in Stewart, J. M. and Young, J., Solid Phase PeptideSynthesis, 2nd Ed., Pierce Chemical Co., Rockford, Ill., (1984), and inRivier et al., U.S. Pat. No. 5,064,939 (Nov. 12, 1991) the disclosure ofthe latter of which is incorporated herein by reference.

A methylbenzhydrylamine resin is used as the solid phase support andfacilitates production of the amidated peptide. Amino acid residues, inthe form of their Boc (tert-butyloxycarbonyl) derivatives, are coupledsuccessively to the resin using dicyclohexylcarbodiimide (DCC) as thecoupling or condensing agent. At each cycle of stepwise amino acidaddition, the Boc group is removed by acidolysis with 50 percent (v/v)trifluoroacetic acid (TFA) in methylene chloride, using an appropriatescavenger, such as 1,2 ethanedithiol, thereby exposing a new α-aminogroup for the subsequent coupling step. More specifically, when anautomated machine and about 5 grams of resin are used, following thecoupling of each amino acid residue, washing, deblocking and coupling ofthe next residue are preferably carried out according to the followingschedule:

MIX TIMES STEP REAGENTS AND OPERATIONS MIN. 1 CH₂Cl₂ wash-40 ml. (2times) 3 2 Methanol (MeOH) wash-30 ml. (2 times) 3 3 CH₂Cl₂ wash-80 ml.(3 times) 3 4 50 percent TFA plus 5 percent 1,2-ethane- 12 dithiol inCH₂Cl₂-70 ml. (2 times) 5 Isopropanol wash-40 ml. (2 times) 3 6 TFA 12.5percent in CH₂Cl₂-70 ml. 5 (2 times) 7 MeOH wash-40 ml. (2 times) 2 8CH₂Cl₂ wash-80 ml. (3 times) 3 9 Boc-amino acid (10 mmoles) in 30 ml. ofeither 30-300 DMF or CH₂Cl₂, depending upon the solubility of theparticular protected amino acid, (1 time) plus DCC (10 mmoles) in CH₂Cl₂

Side chain protecting groups are generally chosen from among thestandard set of moderately acid-stable derivatives. Such protectinggroups are preferably ones that are not removed during deblocking bytrifluoroacetic acid in methylene chloride; however, all are cleavedefficiently by anhydrous hydrofluoric acid (HF) to release thefunctional side chains. Cysteine residues in positions 2, 3, 11, 14, 16and 23 of the peptide are protected by p-methoxy-benzyl (Mob) groups soas to expose sulfhydryls upon deprotection. The phenolic hydroxyl groupof Tyr is protected by 2-bromo-benzyloxycarbonyl (Brz). The side chainof 4-hydroxyproline (4Hyp) is protected by benzyl ether (OBzl), and itis commercially available in this protected form. The side chain of Argis protected with Tos (p-toluenesulfonyl). The side chain of Asp isprotected as the cyclohexyl ester (OChx), and the primary amino sidechain of Lys is protected with 2-chlorobenzyloxycarbonyl (Clz). Theimidazole nitrogen of His is protected by Tos. Serine is protected bybenzyl ether (OBzl). Asn is coupled without side chain protection in thepresence of hydroxybenzotriazole (HOBt).

At the end of the synthesis, the following peptide intermediate isobtained:Boc-Gly-Cys(Mob)-Cys(Mob)-Gly-Ser(OBzl)-Tyr(Brz)-Pro-Asn-Ala-Ala-Cys(Mob)-His(Tos)-Pro-Cys(Mob)-Ser(OBzl)-Cys(Mob)-Lys(Clz)-Asp(OChx)-Arg(TOS-4Hyp(Bzl)-Ser(OBzl)-Tyr(Brz)-Cys(Mob)-Gly-Gla-MBHAresin support. All the side-chain blocking groups are HF-cleavable.

After removing the N-terminal Boc group with TFA, the linear peptide iscleaved from the resin and deprotected with HF, using 150 milliliters ofHF, 16 ml of anisole and about 4 ml dimethyl sulfide for about 1.5 hoursat 0° C., which removes all the remaining protecting groups. Anyvolatiles are removed by the application of a vacuum, and the peptide iswashed with ethylether and then dissolved in 5 percent acetic acid. Thesolution is then diluted to about 15 liters and pH is adjusted to about8.0 with diisopropyl ethylamine. It is exposed to air-oxidation is acold room at about 4° C. for 4 days to form the disulfide cross links orbridges. One drop of mixture is recovered about every 12 hours and addedto one drop of a solution containing dithio-bis(2-nitrobenzoic) acid ina molar buffer of K₂HPO₄ (pH 8) in order to follow the progress of theoxidation reaction (Ellman test). During the whole reaction, the pH wasmaintained at 8 by addition of diisopropylethylamine. After 50 hours,the absence of yellow coloration is observed in the test withdithio-bis(2-nitrobenzoic acid).

After formation of the disulfide bridges, the cyclized pool of peptidesis applied to a Bio-Rex-70 column (5×15 cm), washed in distilled water(100 ml), and eluted with 50% acetic acid. The cyclized peptidefractions are collected and lyophilized.

The lyophilized peptide fractions are then purified by preparative orsemi-preparative HPLC as described in Rivier, et al., J. Chromatography,288, 303-328 (1984); and Hoeger, et al., BioChromatography, 2, 3,134-142 (1987). The chromatographic fractions are carefully monitored byHPLC, and only the fractions showing substantial purity are pooled.

The peptide is judged to be homogeneous by reversed-phase highperformance liquid chromatography using a Waters HPLC system with a0.46×25 cm. column packed with 5 μm C₁₈ silica, 300 Å pore size. Thedetermination is run at room temperature using gradient conditions with2 buffers. Buffer A is an aqueous trifluoroacetic acid (TFA) solutionconsisting of 1.0 ml of TFA per 1000 ml. of solution. Buffer B is 1 mlTFA diluted to 400 ml with H₂O which is added to 600 ml. ofacetonitrile. The analytical HPLC was run under gradient conditionswhich vary uniformly from 20 volume percent (v/o) Buffer B to 35 v/oBuffer B over 10 minutes, at a constant flow rate of 2 ml. per minute;the retention time for the biologically active cyclic conotoxin is 10.6minutes.

The product is also characterized by amino acid analysis and by toxicitytests. One microgram of the synthetic toxin injected intracerebrally(IC) in a mouse is lethal in less than 10 minutes showing that thesynthetic product is highly toxic, and thus synthesis by the describedmethod, if followed by air-oxidation, achieves the correct disulfidepairing arrangement to assure biological activity. The synthetic peptideis shown to be substantially identical with the native conotoxin as aresult of coelution on HPLC, amino acid analysis and biologicalactivity. This peptide binds to and inhibits the function of theacetylcholine receptor, thereby causing paralysis and thereafter death.It can be used in assays for the acetylcholine receptor.

EXAMPLE 2

Conotoxin SEQ ID NO:2 (also referred to as J-005), having the chemicalformula:H-pGlu-Lys-Ser-Leu-Val-Pro-Ser-Val-Ile-Thr-Thr-Cys-Cys-Gly-Tyr-Asp-4Hyp-Gly-Thr-Met-Cys-4Hyp-4Hyp-Cys-Arg-Cys-Thr-Asn-Ser-Cys-NH₂is synthesized by stepwise elongation from the carboxyl terminus, usingthe solid phase synthesis procedure as set forth in Example 1 and thesame methyl benzyhydrylamine resin.

The side chains of hydroxyproline, threonine and serine are protected bybenzyl ether (Bzl).

At the end of the synthesis, the following peptide intermediate isobtained:Boc-pGlu-Lys(Clz)-Ser(Bzl)-Leu-Val-Pro-Ser(Bzl)-Val-Ile-Thr(Bzl)-Thr(Bzl)-Cys(Mob)-Cys(Mob)-Gly-Tyr(Brz)-Asp(OChx)-4Hyp(Bzl)-Gly-Thr(Bzl)-Met-Cys(Mob)-4Hyp(Bzl)-4Hyp(Bzl)-Cys(Mob)-Arg(Tos)-Cys(Mob)-Thr(Bzl)-Asn-Ser(Bzl)-Cys(Mob)-MBHAresin support. All the side-chain blocking groups are HF-cleavable.

After removing the N-terminal Boc group with TFA, the linear peptide iscleaved from 3 grams of the resin and deprotected, using 100 millilitersof HF, 1 ml of anisole and about 4 ml dimethyl sulfide for about 1.5hours at 0° C., which removes all the remaining protecting groups. Anyvolatiles are removed by the application of a vacuum, and the peptide iswashed with ethylether and then extracted with 10 percent acetic acidcontaining 10% cyanomethane. The solution is then diluted to about 4liters and a pH of about 6.95. The solution is exposed to air-oxidationin a cold room at about 4° C. for a time sufficient to completelyoxidize by forming disulfide crosslinks or bridges, i.e., a period ofabout 1 to 2 weeks.

After formation of the disulfide bridges, the cyclized pool of peptidesis applied to a Bio-Rex-70 column (5×15 cm) an eluted with 50% aceticacid. The cyclized peptide fractions are collected and lyophilized. Thesynthetic peptide is shown to be substantially identical with the nativeconotoxin as a result of coelution on HPLC, amino acid analysis andbiological activity, which comparison is made with the native conotoxinfollowing deglycosylation to remove the carbohydrate linked to Ser inthe 7-position which increases bioactivity.

When injected IC into mice, the peptide causes mice to become spasticand to suffer paralysis. It is thus known to have high affinity andspecificity for a particular receptor and can be used to target thisreceptor and in assays for this receptor.

EXAMPLE 3

The peptide OB-34 (SEQ ID NO:3) is produced by using the synthesis asgenerally set forth in Example 1. The peptide in question has thefollowing formula:H-Cys-Cys-Gly-Val-4Hyp-Asn-Ala-Ala-Cys-Pro-4Hyp-Cys-Val-Cys-Asn-Lys-Thr-Cys-Gly-NH₂

The synthesis is carried out on an MBHA resin, and Boc is used toprotect the α-amino groups. The same side chain protecting units areused as described hereinbefore.

About 4½ grams of the peptide-resin is treated with 5 milliliters ofanisole, 1 milliliter of methylethyl sulfide, and 60 milliliters of HFfor ½ hour at −20° C. and 1 hour at 0° C. The peptide is then extractedand dissolved in 4.5 liters of ammonium acetate buffer, a solutioncontaining about 10 grams of ammonium acetate at a pH of about 4.3 pH isadjusted to about 7.75 with ammonium hydroxide, and the solution ismaintained in a cold room at about 4° C. for a sufficient length of timeto allow complete air-oxidation to occur. Purification is then carriedout as previously described with respect to Example 2, and the purifiedpeptide is subjected to analytical HPLC. It is found to exhibit a singlepeak with both a gradient flow and with isocratic flow of appropriatebuffers. The purity of the compound was estimated to be greater thanabout 99 percent. The synthetic peptide coelutes with the native peptideon HPLC.

Injection of 1 microgram of the synthetic peptide OB-34 intracerebrallyinto a mouse shows that the mouse exhibits a reproducible physicaleffect indicative of binding to a specific receptor and confirms thatthe air-oxidation produces appropriate cross-linking so that thesynthetic conotoxin exhibits biological potency. It is thus known tohave high affinity and specificity for a particular receptor and can beused to target this receptor and in assays for this receptor.

EXAMPLE 4

The peptide J--019 (SEQ ID NO:4) is synthesized using the procedure asdescribed with respect to Example 1. The synthetic peptide has thefollowing formula:H-Gly-Cys-Cys-Gly-Ser-Tyr-4Hyp-Asn-Ala-Ala-Cys-His-4Hyp-Cys-Ser-Cys-Lys-Asp-Arg-4Hyp-Ser-Tyr-Cys-Gly-Gln-NH₂

An MBHA resin is used, and Boc is used to protect the α-amino groups ofeach of the amino acids employed in the synthesis. Side chain protectinggroups as set forth with respect to Example 1 are similarly employed.Cleavage from the resin and air-oxidation to carry out cyclicization areperformed as set forth in Example 1.

The cyclic peptide is purified using the procedure set forth in Example1 and checked for purity via analytical HPLC, which shows that asubstantially pure synthetic material is obtained. The synthetic peptideis shown to be substantially identical with the native conotoxin as aresult of coelution on HPLC, amino acid analysis and biologicalactivity. Injection of the peptide intracerebrally into a mouse shows aninitial attack of violent scratching followed by paralysis and ultimatedeath, confirming that air-oxidation can produce appropriatecross-linking so that the synthetic conotoxin exhibits biologicalpotency. It is thus known to have high affinity and specificity for aparticular receptor and can be used to target this receptor and inassays for this receptor.

EXAMPLE 5

The procedure of Example 4 is repeated with a single change of the aminoacid in the 13-position to substitute proline for 4-hydroxyproline andthereby synthesize the peptide J-026 (SEQ ID NO:5). The syntheticpeptide has the following formula:H-Gly-Cys-Cys-Gly-Ser-Tyr-4Hyp-Asn-Ala-Ala-Cys-His-Pro-Cys-Ser-Cys-Lys-Asp-Arg-4Hyp-Ser-Tyr-Cys-Gly-Gln-NH₂

Cleavage from the resin and air-oxidation to carry out cyclicization areperformed as set forth in Example 1.

Analytical HPLC shows the substantially pure compound is obtained. Thesynthetic peptide is shown to be substantially identical with the nativeconotoxin as a result of coelution on HPLC, amino acid analysis andbiological activity. Testing by IC injection into a mouse gives asimilar biological result to that obtained in Example 10, i.e., violentscratching followed by paralysis and death. It is thus known to havehigh affinity and specificity for a particular receptor and can be usedto target this receptor and in assays for this receptor.

EXAMPLE 6

The synthesis of peptide OB-26 (SEQ ID NO:6) is carried out using aprocedure generally the same as that described with respect to Examples1 and 3. The synthetic peptide has the following formula:H-Cys-Cys-Gly-Val-4Hyp-Asn-Ala-Ala-Cys-His-4Hyp-Cys-Val-Cys-Lys-Asn-Thr-Cys-NH₂

Cleavage from the MBHA resin and air-oxidation are carried out as setforth in Example 1. HPLC purification of the cross-linked peptide iscarried out in a similar manner. The resultant synthetic peptide ischecked by analytical HPLC and shown to constitute a substantially purecompound. The synthetic peptide is shown to be substantially identicalwith the native conotoxin as a result of coelution on HPLC, amino acidanalysis and biological activity. Injection of 1 microgram of thesynthetic peptide IC into a mouse results in a reproducible physicaleffect, which verifies that the appropriate disulfide linkages areachieved during the air-oxidation step. It is believed that the peptidehas high affinity and specificity for a particular receptor and that itcan be used to target this receptor and to assay for this receptor.

EXAMPLE 7

Synthesis of the peptide J-029 (SEQ ID NO:7) is carried out on achloromethylated resin in the same general manner as set forth inExample 6. The synthetic peptide has the following formula:H-Gly-4Hyp-Ser-Phe-Cys-Lys-Ala-Asp-Glu-Lys-4Hyp-Cys-Glu-Tyr-His-Ala-Asp-Cys-Cys-Asn-Cys-Cys-Leu-Ser-Gly-Ile-Cys-Ala-Hyp-Ser-Thr-Asn-Trp-Ile-Leu-Pro-Gly-Cys-Ser-Thr-Ser-Ser-Phe-Phe-Lys-Ile-OH

The peptide is cleaved from the resin with anisole, methylethyl sulfideand HF, and air-oxidation is then carried out under the condition asgenerally set forth in Example 1 in order to obtain the cyclic compound.Thereafter, purification is carried out using HPLC as set forthhereinbefore. Ultimate subjection of the purified peptide to analyticalHPLC shows that a substantially pure compound is obtained. The syntheticpeptide is shown to be substantially identical with the native conotoxinas a result of coelution on HPLC, amino acid analysis and biologicalactivity.

Injection of a dose of about 1 microgram of the synthetic conotoxin ICinto a mouse shows substantially immediate paralysis occurring. It isthus known to have high affinity and specificity for a particularreceptor and can be used to target this receptor and in assays for thisreceptor.

EXAMPLE 8

The synthesis of peptide OB-20 (SEQ ID NO:8) having the formula:H-Gly-Cys-Cys-Ser-His-Pro-Ala-Cys-Ser-Gly-Lys-Tyr-Gln-Gla-Tyr-Cys-Arg-Gla-Ser-NH₂is carried out generally as set forth in Example 3 using an Fmocstrategy on a 2,4-dimethoxy-alkoxybenzyl amine resin.

The peptide is cleaved from the resin using a mixture of TFA,thioanisole, water and DCM in the following volume ratios: 40:10:1:44.Cleavage is carried out for about 8 hours at 37° C. Following cleavage,air-oxidation is carried out to cyclize the peptide as set forth inExample 1.

Purification of the cyclized peptide is carried out as set forthhereinbefore. Subjection of the purified peptide to HPLC and amino acidanalysis shows that a peptide having a purity of greater than 95 percentis obtained, which has the expected ratio of residues when subjected toamino acid analysis. The synthetic peptide coelutes with the nativepeptide on HPLC.

Injection of 1 microgram of the synthetic peptide OB-20 intracerebrallyinto a mouse shows that the mouse exhibits a reproducible physicaleffect and confirms that air-oxidation produces appropriatecross-linking so that the synthetic conotoxin exhibits biologicalpotency. It is thus known to have high affinity and specificity for aparticular receptor and can be used to target this receptor and inassays for this receptor.

EXAMPLE 9

A synthesis, as generally set forth in Example 1, is carried out usingabout 25 grams of a chloromethylated polystyrene resin of the typegenerally commercially available to produce peptide J-021 (SEQ ID NO:9)which has the following formula:H-His-4Hyp-4Hyp-Cys-Cys-Leu-Tyr-Gly-Lys-Cys-Arg-Arg-Tyr-4Hyp-Gly-Cys-Ser-Ser-Ala-Ser-Cys-Cys-Gln-OH.

Similar side chain protecting groups are provided as described inExample 1, and the hydroxyl side chain of 4-hydroxyproline is protectedas the benzyl ether. Coupling of the N-terminal His residue is carriedout using Boc-His (Tos) dissolved in DMF and using about 3 millimoles ofbenzotriazol-1-yl-oxy-tris(dimethylamino)phosphonium hexafluorophosphate(BOP) as a coupling agent.

After the final His residue is coupled to the peptide-resin, the Bocgroup is removed using 45 percent TFA in methylene chloride. Thepeptide-resin is then treated with anisole and methylethyl sulfide andHF. Five grams of resin are treated with 10 milliliters of anisole, oneml of methylethyl sulfide and 125 ml of HF for ½ hour at −20° C. and 1hour at 0° C. The cleaved peptide is then extracted using 200milliliters of 50 percent acetic acid at a temperature below 0° C.Thereafter, the extracted peptide is dissolved in 8 liters of 1 percentammonium acetate at a pH of about 4.35. The pH is raised to about 7.74with ammonium hydroxide, and air-oxidation is effected as described inExample 1.

Purification is carried out as described in Example 1, and then purityis checked using analytical HPLC. The peptide is applied to a reversedphase C₁₈ column, and then eluted by subjecting the column to a gradientof buffers A and B at a flow rate of about 0.21 milliliters per minute,which gradient changes uniformly from 0 percent buffer B to 20 percentbuffer B over a time period of 20 minutes. Buffer A is a 1 percentaqueous solution of TFA, and buffer B is 0.1% TFA and 70% acetonitrile.This HPLC shows that the peptide elutes at about 18.6 minutes and has apurity of greater than 99 percent. The synthetic peptide coelutes withthe native peptide on HPLC. Amino acid analysis of the pure peptideshows that the expected residues are obtained.

It is believed that testing will show this peptide to have high affinityand specificity for a particular receptor so that it can be used totarget this receptor or to assay for this receptor.

EXAMPLE 10

The peptide J-010 (SEQ ID NO:10) is synthesized using the procedure asgenerally set forth with respect to Example 8 using an Fmoc protectionstrategy. The synthetic peptide has the following formula:H-Cys-Lys-Thr-Tyr-Ser-Lys-Tyr-Cys-Gla-Ala-Asp-Ser-Gla-Cys-Cys-Thr-Gla-Gln-Cys-Val-Arg-Ser-Tyr-Cys-Thr-Leu-Phe-NH₂.

The peptide is cleaved from the resin using a mixture of TFA,thioanisole, water and DCM in the following volume ratios: 40:10:1:44.Cleavage is carried out for about 8 hours at 37° C. Following cleavage,air-oxidation is carried out to cyclize the peptide as previouslydescribed.

Purification of the cyclized peptide is carried out as set forthhereinbefore, and subjection of the purified peptide to HPLC shows thata substantially pure peptide is obtained. The synthetic peptide is shownto be substantially identical with the native conotoxin as a result ofcoelution on HPLC, amino acid analysis and biological activity.Injection of about 1 microgram of the synthetic peptide intracerebrallyinto a mouse shows that the mouse begins rapid running and stretching,ultimately resulting in death. It is thus known to have high affinityand specificity for a particular receptor and can be used to target thisreceptor and in assays for this receptor.

EXAMPLE 11

A synthesis as generally performed in Example 1 is carried out toproduce peptide J-008 (SEQ ID NO:11) having the formula:H-Ser-Thr-Ser-Cys-Met-Glu-Ala-Gly-Ser-Thr-Cys-Gly-Ser-Thr-Thr-Arg-Ile-Cys-Cys-Gly-Tyr-Cys-Ala-Tyr-Phe-Gly-Lys-Lys-Cys-Ile-Asp-Tyr-Pro-Ser-Asn-OH.

The C-terminal residue in the peptide is Asn in its free acid form. AnMBHA resin was used along with the incorporation of Boc-protected Aspthrough its β-carboxylic group.

Cleavage from the resin and cyclization is carried out as in Example 1.The final product is similarly purified to homogeneity by HPLC, andamino acid analysis of the purified peptide gives the expected results.The synthetic peptide coelutes with the native peptide on HPLC.

The synthetic toxin is injected IC into a mouse, and it proves lethal inless than 10 minutes, confirming that the synthetic product is highlytoxic and that the stated synthesis produces a compound havingbiological activity. It is thus known to have high affinity andspecificity for a particular receptor and can be used to target thisreceptor and in assays for this receptor.

EXAMPLE 12

Synthesis of conotoxin SEQ ID NO:12 (also referred to as J-017), havingthe formula:H-Gly-Glu-Gla-Gla-Val-Ala-Lys-Met-Ala-Ala-Gla-Leu-Ala-Arg-Gla-Asn-Ile-Ala-Lys-Gly-Cys-Lys-Val-Asn-Cys-Tyr-Pro-OHis carried out generally similarly to that of Example 1 but using themodifications described hereinafter.

A commercially available p-alkoxybenzyl alcohol resin is used for thesynthesis, which is a standard resin used in solid phase synthesesemploying the Fmoc-amino acid strategy. Fluorenylmethyloxycarbonyl(Fmoc) is used to protect the α-amino groups of each of the amino acids,and Boc protection is used for the side-chain amino groups of Lys. TheTyr side chain is protected by O-tBu, and the Cys side chain isprotected by diphenylmethyl (trityl). The carboxyl side chain of Glu andthe side chains of Gla are protected by O-t-Bu as described hereinafter.Arg is protected by 4-methoxy-2,3,6-trimethylbenzenesulfonyl (Mtr).

Fmoc-L-Gla(O-t-Bu)₂-OH is prepared as set forth hereinafter.Condensation of Z-L-Ser(Tos)-OCH₃ with di-tert-butyl malonate, to giveZ-DL-Gla(O-t-Bu)₂-OCH₃ is carried out by a modification of the procedureof Rivier et al. Biochemistry 26, 8508-8512 (1987). Sodium hydride isrinsed twice with pentane, suspended in absolute benzene, and then addedto the benzene solution of di-tert-butyl malonate. The reaction isallowed to proceed to completion with 10 minutes of reflux. Theresulting suspension is cooled in an ice bath, and the Z-L-Ser(Tos)-OCH₃dissolved in benzene/tetrahydrofuran is added under an argon atmospherewith vigorous stirring and continued cooling at 0° C. for 2 hours.Stirring is maintained for additional 48 hours at room temperature. Atthis time, the suspension is cooled and washed successively with icewater. 1N HCl, and water. After rotary evaporation at room temperature,the oil is dissolved in benzene, and pentane is added to initiatecrystallization. The yield is 40-60% for a preparation of 0.5 mole. Themethyl ester is hydrolyzed by dissolving in alcohol and adding 1,2 equivof KOH dissolved in water/ethanol. The solution is allowed to remain atroom temperature for several days; the reaction is monitored by HPLCusing a C₁₈ 5-μm column, with 0.1% TFA-acetonitrile as the solvent. Whenthe reaction is complete, the solution is evaporated at roomtemperature, and the product extracted with ethyl acetate after theaddition of NaHSO₄. The ethyl acetate extract is dried over Na₂SO₄ andevaporated under reduced pressure; the yield is 80-90%.

The D- and L-isomers are resolved by crystallization of the quinine saltof the D-isomer. Z-DL-di-t-Bu-Gla-OH in ethyl acetate is reacted with anequivalent amount of quinine. The crystals are separated from the motherliquid, and the Z-D-di-t-Bu-Gla-OH is recrystallized from ethyl acetate.The quinine salt is suspended in ether, and quinine is removed by theaddition of a 20% citric acid solution at 0° C. The same process is usedto remove quinine from the liquid phase. The L-isomer is precipitated inthe form of its ephedrine salt from ethyl acetate-pentane andrecrystallized (Marki et al., Helv. Chim. Acta. 60, 798-800, 1977).Elimination of ephedrine by acid extraction, hydrogenation of the Zgroup, and introduction of the Fmoc are all standard laboratoryprocedures. Optical purity of the L- and D-isomers ofFmoc-Gla(O-t-Bu)₂-OH is assessed after hydrolysis to Glu (6N HCl, 110°C., 20 hours), and each is approximately 99% pure.

The coupling of the Fmoc-protected amino acids to the resin isaccomplished using a schedule generally similar to that set forth inExample 1 but removing the Fmoc group via the use of a 20 percentsolution (v/v) of freshly distilled piperidine in dimethylformamide(DMF) for 10 minutes. Thorough resin washing is accomplished by repeatedapplication of DMF, methanol, or dichloromethane (DCM). Couplings aremediated by DCC in either DCM, DMF, or mixtures thereof, depending uponthe solubility of the particular amino acid derivative. Fmoc-Asn isincorporated into the peptide with an unprotected side chain, in thepresence of 2 equiv of HOBT, and is coupled in DMSO/DMF or DMSO/DCM.

The peptide is released from 4 grams of the peptide resin as theC-terminal free acid by treatment with a freshly prepared mixture ofTFA, thioanisole, H₂O, EDT and DCM (40/18/1/2/49) (40 ml) at about 37°C. for 6-8 hours. Trial cleavages on small amounts demonstrate that thepeptide is freed and that all side-chain protection, including thedifficult Mtr group, are removed while Gla remains intact.

The peptide is precipitated from the cleavage solution after extractionwith methyl tert-butyl ether. The peptide is then dissolved in distilledwater, the pH of the resulting solution is adjusted to approximately 7-8with dilute ammonium hydroxide, after separating the resin byfiltration. Formation of the disulfide cross-link is carried out on thecrude peptide product using a liquid phase, air-oxidation step in a coldroom as described with respect to Example 1. The crude peptide ispurified by preparative HPLC using a preparative cartridge (15-20 μm,300 Å Vydac C₁₈) and a TEAP buffer, pH 2.25, and also with a 0.1% TFAbuffer using appropriate gradients of acetonitrile. Highly purifiedfractions are pooled and lyophilized, yielding peptide as its TFA salt.Optical rotation in 1% acetic acid measures [α]_(D)=−64° (c=1) at 20° C.Amino acid analysis gives the expected values. FAB mass spectrometry isperformed on the peptide, and the spectrum shows a protonated molecularion (MH⁺) at m/z=3097.4 corresponding to the calculated monoisotopicpeptide of 3097.36. A chromatogram of the crude preparation after TFAcleavage and deprotection illustrates that the major product isparticularly pure and that only a relatively small amount of hydrophobicimpurities are present. Sequence analysis gives the expected residue ateach cycle, except for blanks with Gla residues, confirming that thepure target peptide is obtained. The synthetic peptide coelutes with thenative peptide on HPLC.

When injected IC into young mice, it causes sleeping; however, wheninjected into older mice, it causes hyperactivity. It is thus known tohave high affinity and specificity for a particular receptor and can beused to target this receptor and in assays for this receptor,tentatively identified as the NMDA receptor.

EXAMPLE 13

A synthesis of the linear peptide J-004 (SEQ ID NO:13) is carried out onan MBHA resin using the procedure as generally set forth in Example 1.The linear peptide J-004 has the following formula:H-Glu-Ser-Glu-Glu-Gly-Gly-Ser-Asn-Ala-Thr-Lys-Lys-Pro-Tyr-Ile-Leu-NH₂.

The ultimate linear peptide is purified and subjected to amino acidanalysis; it shows that the expected residues are obtained in thepeptide sequence. The synthetic peptide coelutes with the native peptideon HPLC, after the native conotoxin has been deglycosylated to removethe carbohydrate which is linked to Thr in the 10-position which appearsto increase bioactivity. Testing of the synthetic peptide by injectionIC into a mouse shows that the mouse quickly becomes sluggish and unableto stand or function normally, which demonstrates that the syntheticpeptide has the expected biological potency. It is thus known to havehigh affinity and specificity for a particular receptor and can be usedto target this receptor and in assays for this receptor.

These synthetic peptides, for administration to humans, should have apurity of at least about 95 percent (herein referred to a substantiallypure), and preferably have a purity of at least about 98 percent. Purityfor purposes of this application refers to the weight of the intendedpeptide as compared to the weight of all peptide fragments present.These synthetic peptides, either in the free form or in the form of anontoxic salt, are commonly combined with a pharmaceutically orveterinarily acceptable carrier to create a composition foradministration to animals, including humans, or for use in in vitroassays. In vivo administration should be carried out by a physician andthe required dosage will vary with the particular objective beingpursued. In this respect, guidelines have been developed for the use ofother conotoxins such as conotoxin GI and such are well known in thisart are employed for the particular purpose of use.

As indicated hereinbefore, DNA encoding the amino acid structure of anyof these conotoxins can be used to produce the proteins recombinantly aswell as to afford different varieties of plants with pesticidalproperties.

To synthesize a protein having the desired conotoxin amino acid residuesequence by recombinant DNA, a double-stranded DNA chain which encodesthe sequence might be synthetically constructed. Although it is nowadaysfelt that PCR techniques would be the method of choice to produce DNAchains, a DNA chain encoding the desired sequence could be designedusing certain particular codons that are more efficient for polypeptideexpression in a certain type of organism, i.e. selection might employthose codons which are most efficient for expression in the type oforganism which is to serve as the host for the recombinant vector.However, any correct set of codons will encode a desired product,although perhaps slightly less efficiently. Codon selection may alsodepend upon vector construction considerations; for example, it may benecessary to avoid placing a particular restriction site in the DNAchain if, subsequent to inserting the synthetic DNA chain, the vector isto be manipulated using the restriction enzyme that cleaves at such asite. Also, one should of course avoid placing restriction sites in theDNA chain if the host organism, which is to be transformed with therecombinant vector containing the DNA chain, is known to produce arestriction enzyme that would cleave at such a site within the DNAchain.

To assemble such a synthetic, nonchromosomal, conotoxin-encoding DNAchain, oligonucleotides are constructed by conventional procedures suchas those described in J. Sambrook et al., Molecular Cloning, ALaboratory Manual, Cold Spring Harbor Laboratory Press, New York (1989)(hereinafter, Sambrook et al.). Sense and antisense oligonucleotidechains, up to about 70 nucleotide residues long, are synthesized,preferably on automated synthesizers, such as the Applied Biosystem Inc.Model 380A DNA synthesizer. The oligonucleotide chains are constructedso that portions of the sense and antisense oligonucleotides overlap,associating with each other through hydrogen bonding betweencomplementary base pairs and thereby forming double stranded chains, inmost cases with gaps in the strands. Subsequently, the gaps in thestrands are filled in, and oligonucleotides of each strand are joinedend to end with nucleotide triphosphates in the presence of appropriateDNA polymerases and/or with ligases.

As an alternative to such stepwise construction of a synthetic DNAchain, the cDNA corresponding to the desired conotoxin may be cloned. Asis well known, a cDNA library or an expression library is produced in aconventional manner by reverse transcription from messenger RNA (mRNA)from suitable tissue from the cone snail of interest. To select clonescontaining desired sequences, a hybridization probe or a mixed set ofprobes which accommodate the degeneracy of the genetic code andcorrespond to a selected portion of the protein of interest are producedand used to identify clones containing such sequences. Screening of suchan expression library with antibodies made against the protein may alsobe used, either alone or in conjunction with hybridization probing, toidentify or confirm the presence of DNA sequences in cDNA library cloneswhich are expressing the protein of interest. Such techniques aretaught, for example in Sambrook et al., supra.

In addition to the protein-encoding sequences, a DNA chain shouldcontain additional sequences depending upon vector constructionconsiderations. Typically, a synthesized DNA chain has linkers at itsends to facilitate insertion into restriction sites within a cloningvector. A DNA chain may be constructed so as to encode the protein aminoacid sequences as a portion of a fusion polypeptide; and if so, it willgenerally contain terminal sequences that encode amino acid residuesequences that serve as proteolytic processing sites, whereby thedesired polypeptide may be proteolytically cleaved from the remainder ofthe fusion polypeptide. The terminal portions of the synthetic DNA chainmay also contain appropriate start and stop signals.

Accordingly, a double-stranded conotoxin-encoding DNA chain isconstructed or modified with appropriate linkers for its insertion intoa particular appropriate cloning vectors. The cloning vector that is tobe recombined to incorporate the DNA chain is selected appropriate toits viability and expression in a host organism or cell line, and themanner of insertion of the DNA chain depends upon factors particular tothe host. For example, if the DNA chain is to be inserted into a vectorfor insertion into a prokaryotic cell, such as E. coli, the DNA chainwill be inserted 3′ of a promoter sequence, a Shine-Delgarno sequence(or ribosome binding site) that is within a 5′ non-translated portionand an ATG start codon. The ATG start codon is appropriately spaced fromthe Shine-Delgarno sequence, and the encoding sequence is placed incorrect reading frame with the ATG start codon. The cloning vector alsoprovides a 3′ non-translated region and a translation termination site.For insertion into a eukaryotic cell, such as a yeast cell or a cellline obtained from a higher animal, the conotoxin-encodingoligonucleotide sequence is appropriately spaced from a capping site andin correct reading frame with an ATG start signal. The cloning vectoralso provides a 3′ non-translated region and a translation terminationsite.

Prokaryotic transformation vectors, such as pBR322, pMB9, Col E1, pCR1,RP4 and lambda-phage, are available for inserting a DNA chain of thelength which encodes conotoxin with substantial assurance of at leastsome expression of the encoded polypeptide. Typically, such vectors areconstructed or modified to have one or more unique restriction sitesappropriately positioned relative to a promoter, such as the lacpromoter. The DNA chain may be inserted with appropriate linkers intosuch a restriction site, with substantial assurance of production ofdesired protein in a prokaryotic cell line transformed with therecombinant vector. To assure proper reading frame, linkers of variouslengths may be provided at the ends of the protein-encoding sequences.Alternatively, cassettes, which include sequences, such as the 5′ regionof the lac Z gene (including the operator, promoter, transcription startsite. Shine-Delgarno sequence and translation initiation signal), theregulatory region from the tryptophane gene (trp operator, promoter,ribosome binding site and translation initiator), and a fusion genecontaining these two promoters called the trp-lac or commonly called theTac promoter are available into which the synthetic DNA chain may beconveniently inserted and then the cassette inserted into a cloningvector of choice.

Similarly, eukaryotic transformation vectors, such as, the cloned bovinepapilloma virus genome, the cloned genomes of the murine retroviruses,and eukaryotic cassettes, such as the pSV-2 gpt system (described byMulligan and Berg, Nature 277, 108-114, 1979), the Okayama-Berg cloningsystem (Mol. Cell Biol. 2, 161-170, 1982), and the expression cloningvector recently described by Genetics Institute (Science 228, 810-815,1985), are available which provide substantial assurance of at leastsome expression of conotoxin in the transformed eukaryotic cell line.

As previously mentioned, a convenient way to ensure production of aprotein of the length of the conotoxins of interest is to produce theprotein initially as a segment of a gene-encoded fusion protein. In suchcase, the DNA chain is constructed so that the expressed protein hasenzymatic processing sites flanking the conotoxin amino acid residuesequences. A conotoxin-encoding DNA chain may be inserted, for example,into the beta-galactosidase gene for insertion into E. coli, in whichcase, the expressed fusion protein is subsequently cleaved withproteolytic enzymes to release the conotoxin from beta-galactosidasepeptide sequences.

An advantage of inserting the protein-encoding sequence so that thedesired sequence is expressed as a cleavable segment of a fusionprotein, e.g. as the conotoxin sequence fused within thebeta-galactosidase peptide sequence, is that the endogenous protein intowhich the desired conotoxin sequence is inserted is generally renderednon-functional, thereby facilitating selection for vectors encoding thefusion protein.

The conotoxin proteins may also be reproduced in yeast using knownrecombinant DNA techniques. For example, a suitable plasmid, amplifiedin an E. coli clone, is isolated and cleaved with Eco RI and Sal I. Thisdigested plasmid is electrophoresed on an agarose gel allowing for theseparation and recovery of the amplified insert of interest. The insertis inserted into the plasmic pYep, a shuttle vector which can be used totransform both E. coli and Saccharomyces cerevisiae yeast. Insertion ofthe synthetic DNA chain at this point assures that the DNA sequence isunder the control of a promoter, in proper reading frame from an ATGsignal and properly spaced relative to a cap site. The shuttle vector isused to transform URA3, a strain of S. cerevisiae yeast from which theoratate monophosphate decarboxylase gene is deleted.

The transformed yeast is grown in medium to attain log growth. The yeastis separated from its culture medium, and cell lysates are prepared.Pooled cell lysates are determined by RIA to be reactive with antibodyraised against the conotoxin, demonstrating that a protein containingprotein segment is expressed within the yeast cells.

The production of conotoxins can be carried out in both prokaryotic andeukaryotic cell lines to provide protein for biological and therapeuticuse. While conotoxin synthesis is easily demonstrated using eitherbacteria or yeast cell lines, the synthetic genes should be insertablefor expression in cells of higher animals, such as mammalian tumorcells, and in plants. Such mammalian cells may be grown, for example, asperitoneal tumors in host animals, and certain conotoxins may beharvested from the peritoneal fluid. The cloned DNA is insertable intoplant varieties of interest where the plant utilizes it as a plantdefense gene, i.e. it produces sufficient amounts of the pesticide ofinterest to ward off insects or the like that are natural predators tosuch plant species.

Although the above examples demonstrate that conotoxins can besynthesized through recombinant DNA techniques, the examples do notpurport to have maximized conotoxin production. It is expected thatsubsequent selection of more efficient cloning vectors and host celllines will increase the yield, and known gene amplification techniquesfor both eukaryotic and prokaryotic cells may be used to increaseproduction. Secretion of the gene-encoded protein from the host cellline into the culture medium is also considered to be an importantfactor in obtaining certain of the synthetic proteins in largequantities.

Although the invention has been described with regard to certainpreferred embodiments, it should be understood that various changes andmodifications as would be obvious to one having the ordinary skill inthe art may be made without departing from the scope of the inventionwhich is set forth in appended claims. For example, substitution ofvarious of the amino acid residues depicted in the amino acid sequencesby residues known to be equivalent with those residues can be effectedto produce equivalent peptides having similar biological activities.Moreover, it is known that additional substitutions in the amino acidsequence generally throughout the C-terminal portion of the peptide,i.e. within about ⅓ of the length of the conotoxin nearest itsC-terminus, can be effected in order to produce conotoxins havingphylogenetic specificity; thus, such substitutions in this region can becarried out to produce valuable equivalent structures. The C-terminus ofmany of the illustrated peptides is amidated, and the inclusion of asubstituted amide at the C-terminus of such peptides, as describedhereinbefore, is considered to create an equivalent conotoxin.

Particular features of the invention are emphasized in the claims whichfollow.

1. A substantially pure conotoxin which is highly selective for aspecific human receptor, which conotoxin is selected from the groupconsisting of:Gly-Xaa-Ser-Phe-Cys-Lys-Ala-Asp-Glu-Lys-Xaa-Cys-Glu-Tyr-His-Ala-Asp-Cys-Cys-Asn-Cys-Cys-Leu-Ser-Gly-Ile-Cys-Ala-Xaa-Ser-Thr-Asn-Trp-Ile-Leu-Pro-Gly-Cys-Ser-Thr-Ser-Ser-Phe-Phe-Lys-Ile(SEQ ID NO:7) wherein Xaa is 4Hyp;Gly-Cys-Cys-Ser-His-Pro-Ala-Cys-Ser-Gly-Lys-Tyr-Gln-Xaa-Tyr-Cys-Arg-Xaa-Ser(SEQ ID NO:8) wherein Xaa is Gla and the C-terminus is amidated;His-Xaa-Xaa-Cys-Cys-Leu-Tyr-Gly-Lys-Cys-Arg-Arg-Tyr-Xaa-Gly-Cys-Ser-Ser-Ala-Ser-Cys-Cys-Gln(SEQ ID NO:9) wherein Xaa is 4Hyp;Cys-Lys-Thr-Tyr-Ser-Lys-Tyr-Cys-Xaa-Ala-Asp-Ser-Xaa-Cys-Cys-Thr-Xaa-Gln-Cys-Val-Arg-Ser-Tyr-Cys-Thr-Leu-Phe(SEQ ID NO:10) wherein Xaa is Gla and the C-terminus is amidated;Ser-Thr-Ser-Cys-Met-Glu-Ala-Gly-Ser-Tyr-Cys-Gly-Ser-Thr-Thr-Arg-Ile-Cys-Cys-Gly-Tyr-Cys-Ala-Tyr-Phe-Gly-Lys-Lys-Cys-Ile-Asp-Tyr-Pro-Ser-Asn(SEQ ID NO:11);Gly-Glu-Xaa-Xaa-Val-Ala-Lys-Met-Ala-Ala-Xaa-Leu-Ala-Arg-Xaa-Asn-Ile-Ala-Lys-Gly-Cys-Lys-Val-Asn-Cys-Tyr-Pro(SEQ ID NO:12) wherein Xaa is Gla; andGlu-Ser-Glu-Glu-Gly-Gly-Ser-Asn-Ala-Thr-Lys-Lys-Pro-Tyr-Ile-Leu (SEQ IDNO:13), wherein Glu in the 1-position is pGlu and the C-terminus isamidated.
 2. A conotoxin according to claim 1 having the formula:Gly-Xaa-Ser-Phe-Cys-Lys-Ala-Asp-Glu-Lys-Xaa-Cys-Glu-Tyr-His-Ala-Asp-Cys-Cys-Asn-Cys-Cys-Leu-Ser-Gly-Ile-Cys-Ala-Xaa-Ser-Thr-Asn-Trp-Ile-Leu-Pro-Gly-Cys-Ser-Thr-Ser-Ser-Phe-Phe-Lys-Ile(SEQ ID NO:7) wherein Xaa is 4Hyp.
 3. A conotoxin according to claim 1having the formula:Gly-Cys-Cys-Ser-His-Pro-Ala-Cys-Ser-Gly-Lys-Tyr-Gln-Xaa-Tyr-Cys-Arg-Xaa-Ser(SEQ ID NO:8) wherein Xaa is Gla and the C-terminus is amidated.
 4. Aconotoxin according to claim 1 having the formula:His-Xaa-Xaa-Cys-Cys-Leu-Tyr-Gly-Lys-Cys-Arg-Arg-Tyr-Xaa-Gly-Cys-Ser-Ser-Ala-Ser-Cys-Cys-Gln(SEQ ID NO:9) wherein Xaa is 4Hyp.
 5. A conotoxin according to claim 1having the formula:Cys-Lys-Thr-Tyr-Ser-Lys-Tyr-Cys-Xaa-Ala-Asp-Ser-Xaa-Cys-Cys-Thr-Xaa-Gln-Cys-Val-Arg-Ser-Tyr-Cys-Thr-Leu-Phe(SEQ ID NO:10) wherein Xaa is Gla and the C-terminus is amidated.
 6. Aconotoxin according to claim 1 having the formula:Ser-Thr-Ser-Cys-Met-Glu-Ala-Gly-Ser-Tyr-Cys-Gly-Ser-Thr-Thr-Arg-Ile-Cys-Cys-Gly-Tyr-Cys-Ala-Tyr-Phe-Gly-Lys-Lys-Cys-Ile-Asp-Tyr-Pro-Ser-Asn(SEQ ID NO:11).
 7. A conotoxin according to claim 1 having the formula:Gly-Glu-Xaa-Xaa-Val-Ala-Lys-Met-Ala-Ala-Xaa-Leu-Ala-Arg-Xaa-Asn-Ile-Ala-Lys-Gly-Cys-Lys-Val-Asn-Cys-Tyr-Pro(SEQ ID NO:12) wherein Xaa is Gla.
 8. A conotoxin according to claim 1having the formula:Glu-Ser-Glu-Glu-Gly-Gly-Ser-Asn-Ala-Thr-Lys-Lys-Pro-Tyr-Ile-Leu (SEQ IDNO:13), wherein Glu in the 1-position is pGlu and the C-terminus isamidated.
 9. The conotoxin according to claim 8 wherein the Thr residueis glycosylated.
 10. A substantially pure conotoxin having the aminoacid sequenceGlu-Ser-Glu-Glu-Gly-Gly-Ser-Asn-Ala-Thr-Lys-Lys-Pro-Tyr-Ile-Leu (SEQ IDNO:13 ), wherein Glu in the 1 -position is pGlu.
 11. The substantiallypure conotoxin of claim 10, wherein the Thr residue is glycosylated. 12.An isolated conotoxin which is highly selective for a specific humanreceptor, comprising a conotoxin selected from the group consisting of:Gly-Xaa-Ser-Phe-Cys-Lys-Ala-Asp-Glu-Lys-Xaa-Cys-Glu-Tyr-His-Ala-Asp-Cys-Cys-Asn-Cys-Cys-Leu-Ser-Gly-Ile-Cys-Ala-Xaa-Ser-Thr-Asn-Trp-Ile-Leu-Pro-Gly-Cys-Ser-Thr-Ser-Ser-Phe-Phe-Lys-Ile(SEQ ID NO:7 ) wherein Xaa is 4Hyp;Gly-Cys-Cys-Ser-His-Pro-Ala-Cys-Ser-Gly-Lys-Tyr-Gln-Xaa-Tyr-Cys-Arg-Xaa-Ser(SEQ ID NO:8 ) wherein Xaa is Gla and the C-terminus is amidated;His-Xaa-Xaa-Cys-Cys-Leu-Tyr-Gly-Lys-Cys-Arg-Arg-Tyr-Xaa-Gly-Cys-Ser-Ser-Ala-Ser-Cys-Cys-Gln(SEQ ID NO:9 ) wherein Xaa is 4Hyp;Cys-Lys-Thr-Tyr-Ser-Lys-Tyr-Cys-Xaa-Ala-Asp-Ser-Xaa-Cys-Cys-Thr-Xaa-Gln-Cys-Val-Arg-Ser-Tyr-Cys-Thr-Leu-Phe(SEQ ID NO:10 ) wherein Xaa is Gla and the C-terminus is amidated;Ser-Thr-Ser-Cys-Met-Glu-Ala-Gly-Ser-Tyr-Cys-Gly-Ser-Thr-Thr-Arg-Ile-Cys-Cys-Gly-Tyr-Cys-Ala-Tyr-Phe-Gly-Lys-Lys-Cys-Ile-Asp-Tyr-Pro-Ser-Asn(SEQ ID NO:11 );Gly-Glu-Xaa-Xaa-Val-Ala-Lys-Met-Ala-Ala-Xaa-Leu-Ala-Arg-Xaa-Asn-Ile-Ala-Lys-Gly-Cys-Lys-Val-Asn-Cys-Tyr-Pro(SEQ ID NO:12 ) wherein Xaa is Gla; andGlu-Ser-Glu-Glu-Gly-Gly-Ser-Asn-Ala-Thr-Lys-Lys-Pro-Tyr-Ile-Leu (SEQ IDNO:13 ), wherein Glu in the 1 -position is pGlu and the C-terminus isamidated.
 13. The conotoxin according to claim 12 comprising theformula:Gly-Xaa-Ser-Phe-Cys-Lys-Ala-Asp-Glu-Lys-Xaa-Cys-Glu-Tyr-His-Ala-Asp-Cys-Cys-Asn-Cys-Cys-Leu-Ser-Gly-Ile-Cys-Ala-Xaa-Ser-Thr-Asn-Trp-Ile-Leu-Pro-Gly-Cys-Ser-Thr-Ser-Ser-Phe-Phe-Lys-Ile(SEQ ID NO:7 ) wherein Xaa is 4Hyp.
 14. The isolated conotoxin accordingto claim 12 comprising the formula:Gly-Cys-Cys-Ser-His-Pro-Ala-Cys-Ser-Gly-Lys-Tyr-Gln-Xaa-Tyr-Cys-Arg-Xaa-Ser(SEQ ID NO:8 ) wherein Xaa is Gla and the C-terminus is amidated. 15.The isolated conotoxin according to claim 12 comprising the formula:His-Xaa-Xaa-Cys-Cys-Leu-Tyr-Gly-Lys-Cys-Arg-Arg-Tyr-Xaa-Gly-Cys-Ser-Ser-Ala-Ser-Cys-Cys-Gln(SEQ ID NO:9 ) wherein Xaa is 4Hyp.
 16. The isolated conotoxin accordingto claim 12 comprising the formula:Cys-Lys-Thr-Tyr-Ser-Lys-Tyr-Cys-Xaa-Ala-Asp-Ser-Xaa-Cys-Cys-Thr-Xaa-Gln-Cys-Val-Arg-Ser-Tyr-Cys-Thr-Leu-Phe(SEQ ID NO:10 ) wherein Xaa is Gla and the C-terminus is amidated. 17.The isolated conotoxin according to claim 12 comprising the formula:Ser-Thr-Ser-Cys-Met-Glu-Ala-Gly-Ser-Tyr-Cys-Gly-Ser-Thr-Thr-Arg-Ile-Cys-Cys-Gly-Tyr-Cys-Ala-Tyr-Phe-Gly-Lys-Lys-Cys-Ile-Asp-Tyr-Pro-Ser-Asn(SEQ ID NO:11 ).
 18. The isolated conotoxin according to claim 12comprising the formula:Gly-Glu-Xaa-Xaa-Val-Ala-Lys-Met-Ala-Ala-Xaa-Leu-Ala-Arg-Xaa-Asn-Ile-Ala-Lys-Gly-Cys-Lys-Val-Asn-Cys-Tyr-Pro(SEQ ID NO:12 ) wherein Xaa is Gla.
 19. The isolated conotoxin accordingto claim 12 comprising the formula:Glu-Ser-Glu-Glu-Gly-Gly-Ser-Asn-Ala-Thr-Lys-Lys-Pro-Tyr-Ile-Leu (SEQ IDNO:13 ), wherein Glu in the 1 -position is pGlu and the C-terminus isamidated.
 20. The isolated conotoxin according to claim 19 wherein theThr residue is glycosylated.
 21. An isolated conotoxin comprising theamino acid sequenceGlu-Ser-Glu-Glu-Gly-Gly-Ser-Asn-Ala-Thr-Lys-Lys-Pro-Tyr-Ile-Leu (SEQ IDNO:13 ), wherein Glu in the 1 -position is pGlu.
 22. An isolatedconotoxin comprising the amino acid sequenceGlu-Ser-Glu-Glu-Gly-Gly-Ser-Asn-Ala-Thr-Lys-Lys-Pro-Tyr-Ile-Leu (SEQ IDNO:13 ), wherein Glu in the 1 -position is pGlu and the Thr residue isglycosylated.